The present invention relates to formulated medicines for potentiation of the clinical efficacy of beta-lactam antibiotics, both to prevent and treat infectious disease caused by pathogenic bacteria. The formulated medicines of the present invention contain bovine milk lactoferrin and show remarkable potentiating effect on the clinical efficacy of beta-lactam antibiotics, thus more effectively preventing and treating bacterial diseases of mammals, including human beings. The present invention also relates to a method for the prevention and treatment of bacterial diseases in mammals, including human beings.
Lactoferrin is the major glycoprotein present in the granules of mature neutrophils and is deemed to be one of the host-defense factors, being locally released where pathogenic bacteria infect. Lactoferrin also exists in such diverse secretions as milk, tears, saliva, and digestive juices, and is thought to be one of the factors which prevent mammals from being infected by bacterial pathogens. The function of lactoferrin is not clear but is believed to play a similar role to that of lysozyme and secretory immunoglobulin A. The most important role of lactoferrin is considered to be the covering and protecting of mucous membranes which occupy huge areas on the body surface which are always being threatened by the invasive attack of bacterial pathogens.
Therefore, lactoferrin is regarded as a nonspecific barrier against the invasion of pathogenic bacteria. It probably cooperates with other host-defense factors such as phagocytes, lysozyme, complement and immunoglobulins. In a healthy human adult male, five grams of lactoferrin are produced daily (calculated on the basis of the turnover rate of neutrophils). As it is well known, when acute inflammation caused by bacterial infection occurs, the productivity of lactoferrin increases by approximately six fold to 30 g per day. For this reason lactoferrin is deemed to play a very important role in host-defense mechanisms against bacterial infection and its importance is comparable to such host-defense factors as phagocytes, immunoglobulins and lysozyme.
In mammals, milk, especially colostrum, contains a large amount of lactoferrin. The reason why colostrum contains such large amounts is that it probably protects newborn infants from many kinds of infectious pathogens because a fetus grows aseptically in the maternal uterus and never encounters such kinds of bacterial pathogens before birth. They are highly susceptible to infection by such agents because of the immature properties of their host-defense system. Their gastrointestinal system is free from bacteria at birth and the normal intestinal flora is not formed soon after birth. The role of milk lactoferrin is likely, in cooperation with other host-defense factors, to cover intestinal mucosa, thus protecting the intestine from invasion by pathogenic bacteria and to help the newborns to form normal intestinal microflora.
However, it is not clear how and where lactoferrin acts on the surface of mucosa. Although a number of studies have been conducted during the past two decades, the functions and roles of lactoferrin have not yet been clarified. Moreover, it is unknown what kinds of physiological or pharmacological responses occur on the animal level when lactoferrin derived from other animal species is given orally to another animal species. Bovine milk lactoferrin used in this invention is a minor component of whey protein and it has been very expensive when isolated in relatively pure form. Whey, especially cheese whey, is the most abundant natural source of lactoferrin. Cheese whey is a typical byproduct in dairy industry and major portions of it have been wasted for swine feed or direct spray on meadows as fertilizer.
However, owing to the development of new technology, e.g., large scale separation and purification techniques using ultrafiltration membranes and ion exchange column chromatography, such minor but bioactive proteins as lactoferrin can be efficiently extracted and isolated in native form from whey (which contains numerous minor ingredients). Therefore, relatively pure lactoferrin (approximately more than 85% pure) is now available at a reasonable cost. Nevertheless, these advances are worthless without development of a potential usefulness for lactoferrin. Two reasons are considered why the biomedical utilization of lactoferrin has been hindered. The first reason is a possibility of anaphylactic shock since the bovine lactoferrin is non-self for human beings, so that parenteral administration may result in induction of severe anaphylactic shock when repeatedly administered. Therefore, at the present time, the route of administration of bovine lactoferrin has to be strictly limited to oral administration for human beings. The second reason stems from the possibility of the digestibility of lactoferrin by proteinase in the gastrointestinal tract. When bovine lactoferrin was orally taken by mammals, it is useless when it is hydrolyzed in the gastrointestinal tract to constituent amino acids and peptides before reaching the active site.
Recent immunology studies indicate that macromolecules with non-self antigenic determinants are occasionally absorbed through the intestine where suppressor T-cells recognize them as non-self and suppress helper T-cell proliferation, thus leading to inhibition of the immunological responses to the non-self antigens. That is to say, when intact non-self antigen is absorbed though the intestine without changing the structure, a kind of immunological tolerance is induced.
Therefore, as far as the oral route is concerned, bovine lactoferrin never induces anaphylactic shock in humans even if small portions are absorbed through the intestine without changing its complete structure. Thus, the oral route is the safest way to dose antigenic macromolecules. Moreover, bovine lactoferrin molecules are highly resistant to gastrointestinal digestion and, when taken orally, considerable amounts are excreted in intact form in feces.
According to previous studies, lactoferrin is an antibacterial protein present in the granules of neutrophils and its activity is dependent on the degree of iron saturation; the activity of iron-free lactoferrin, apolactoferrin, is the most potent and its activity is gradually lost as the degree of the saturation elevates. However, the present invention has revealed (as shown in table 1) that even apolactoferrin from bovine milk lacks antibacterial activity in vitro against most pathogenic bacteria. Most of the bacteria showed minimum inhibitory concentrations of more than 6,400 .mu.g/ml, the only exception is Streptococcus epidermidis ATCC 13288 whose growth was inhibited in the presence of 800 .mu.g/ml of apolactoferrin; such concentrations are unattainably high at the whole animal level. Therefore, even though the lactoferrins have very weak antibacterial activity in vitro, it is questionable that they exert direct antibacterial activity against bacteria infecting mammals.